The present invention relates to compositions containing quinazolinones. More particularly, the present invention relates to a composition, containing a quinazolinone derivative, useful for the treatment of angiogenic-associated diseases, as well as for the treatment of malignancies, including inhibition of primary tumor growth, tumor progression and metastasis.
Malignancies are characterized by the growth and spread of tumors. A number of factors are important in the progression of this disease. One crucial factor is angiogenesis, a complex process in which capillary blood vessels grow in an ordered sequence of events [J. Folkman and M. Klagsbrun, Science, Vol. 235, pp 442-447 (1987); J. Folkman and Y. Shing, J. Biol. Chem., Vol. 267, pp. 10931-10934 (1992)]. Once a tumor has started, every increase in tumor cell population must be preceded by an increase in new capillaries that converge on the tumor and supply the cells with oxygen and nutrients [J. Folkman, Perspect. in Biol. and Med., Vol 29, p. 10-36 (1985); J. Folkman, J. Natl. Cancer Inst., Vol. 82, pp. 4-6 (1989); N. Weidner, et al., Amer. J. Pathol., Vol. 143, pp. 401-409 (1993)]. Tumors may thus remain harmless and confined to their tissue of origin, as long as an accompanying angiogenic program is prevented from being activated. Since the angiogenesis-dependent step in tumor progression is shared by solid tumors of all etiologies, the ability to inhibit tumor-associated angiogenesis is a most promising approach in combating cancer [M. S. O'Reilly, et al., Cell, Vol. 79, pp. 316-328 (1994)].
A substantial body of experimental evidence supports the hypothesis that tumor angiogenesis is fundamental for the growth and metastasis of solid tumors [J. Folkman, ibid. (1989); N. Weidner, et al., ibid. (1993); M. S. O'Reilly, et al., ibid. (1994); N. Weidner, et al., N. Eng. J. Med., Vol. 324, pp. 1-8 (1991)]. Indeed, the majority of solid tumors are not even clinically detectable until after the occurrence of neovascularization, whose induction in solid tumors is mediated by one or more angiogenic factors [J. Folkman, ibid. (1987); J. Folkman and Y. Shing, ibid. (1992)].
Furthermore, angiogenesis is also important in a number of other pathological processes, including arthritis, psoriasis, diabetic retinopathy, chronic inflammation, scleroderma, hemangioma, retrolental fibroplasia and abnormal capillary proliferation in hemophiliac joints, prolonged menstruation and bleeding, and other disorders of the female reproductive system [J. Folkman, Nature Medicine, Vol 1, p. 27-31, (1995); J. W. Miller, et al., J. Pathol. Vol. 145, pp. 574-584 (1994); A. P. Adamid, et al., Amer. J. Ophthal., Vol. 118, pp. 445-450 (1994); K. Takahashi, at al., J. Clin. Invest., Vol.93, pp.2357-2364 (1994); D. J. Peacock, et al., J. Exp. Med., Vol. 175, pp. 1135-1138 (1992); B. J. Nickoloff, et al., Amer. J. Pathol., Vol. 44, pp. 820-828 (1994); J. Folkman, Steroid Hormones and Uterine Bleeding, N. J. Alexander and C. d'Arcangues, Eds., American Association for the Advancement of Science Press, Washington, D.C., U.S.A., pp. 144-158 (1992)].
Thus, clearly methods of blocking the mechanism of angiogenesis are necessary. The basic mechanism of angiogenesis is as follows. Briefly, when a new capillary sprout grows from the side of the venule, endothelial cells degrade basement membrane, migrate toward an angiogenic source, proliferate, form a lumen, join the tips of two sprouts to generate a capillary loop, and manufacture new basement membrane [J. Folkman, Perspectives in Biology and Medicine, Vol. 29, pp. 1-36 (1985)].
Degradation and remodeling of the ECM are essential processes for the mechanism of angiogenesis. In addition, ECM components synthesized by endothelial cells (i.e., collagens, laminin, thrombospondin, fibronectin and SPARC) function to regulate endothelial cell growth, migration and shape [J. Bischoff, Trends Cell Biol., No. 5, pp. 69-74 (1995)]. Bovine aortic endothelial cells (BAE) undergoing sprouting and tube formation synthesize type I collagen and SPARC. It was proposed that type I collagen may be involved in directing migration and assembly of the BAE cells [M. L. Iruela-Arispe, et al., Lab. Invest., No. 64, pp. 174-186 (1991). It was also found that exogenous type I collagen promoted rapid tube formation by confluent human dermal microvascular endothelial cells [C. J. Jackson and K. L. Jenkins, Exp. Cell Res., No. 192, pp. 319-323 (1991)]. The tubes contained collagen fibrils in the luminal spaces, suggesting that the endothelial cells use the fibrils to fold and align into tube structures.
Furthermore, in order to extend a capillary blood vessel, interactions must occur between ECM components and the surrounding matrix molecules, which provide a scaffold for the ECM components of the new vessel [Brooks, P. C. et al., Cell, Vol 79, p. 1157-1164, (1994)]. Disruption of cell-matrix interactions induced apoptosis in human endothelial cells. It has been demonstrated that integrin α2β3, which has an enhanced expression in angiogenic vascular cells, promotes a survival signal, since inhibitors of this integrin cause unscheduled apoptosis and disintegration of newly formed blood vessels.
In order to treat angiogenesis-related diseases, several inhibitors of the above mechanism of angiogenesis are being studied, including platelet factor 4, the fumagillin derivative AGM 1470, Interferon α2a, thrombospondin, angiostatic steroids, and angiostatin [J. Folkman, ibid., (1995); M. S. O'Reilly, et al., ibid. (1994); V. Castle, et al., J. Clin. Invest., Vol. 87, pp.1183-1888; D. Ingber, et al., Nature, Vol. 348, pp. 555-557]. All of these compounds have disadvantages. For example, endostatin and angiostatin are proteins, so that they have all of the disadvantages of proteins, including the requirement for being administered parenterally. Therefore, a non-protein inhibitor which would selectively block the underlying mechanism of angiogenesis without adversely affecting other physiological functions, and which could be administered by many different routes, would be extremely useful.
In addition, many of the compounds that are now being evaluated as antiangiogenic agents are proteins, e.g., antibodies, thrombospondin, angiostatin, platelet factor IV [J. Folkman, ibid. (1995); M. S. O'Reilly, et al., ibid. (1994); V. Castle, et al., ibid., P. C. Brooks, et al., ibid. (1994)], which suffer from poor bioavailability and are readily degraded in the body. Hence, these substances should be administered in high doses and frequencies.
Other approaches for cancer treatment focus on cytotoxic therapies, such as chemotherapy or radiation treatments, in order to kill actively proliferating cells. Unfortunately, these therapies are highly toxic to non-cancer cells and cause severe side effects, such as bone marrow suppresssion, hair loss and gastrointestinal disturbances.
As noted above, degradation and remodeling of the ECM are essential processes for the mechanism of angiogenesis. Such processes involve the synthesis of a number of components of the ECM, such as collagen. The synthesis of collagen is also involved in a number of other pathological conditions. For example, clinical conditions and disorders associated with primary or secondary fibrosis, such as systemic sclerosis, graft-versus-host disease (GVHD), pulmonary and hepatic fibrosis and a large variety of autoimmune disorders, are distinguished by excessive production of connective tissue, which results in the destruction of normal tissue architecture and function. These diseases can best be interpreted in terms of perturbations in cellular functions, a major manifestation of which is excessive collagen synthesis and deposition. The crucial role of collagen in fibrosis has prompted attempts to develop drugs that inhibit its accumulation [K. J. Kivirikko, Annals of Medicine, Vol.25, pp. 113-126 (1993)].
Such drugs can act by modulating the synthesis of the procollagen polypeptide chains, or by inhibiting specific post-translational events, which will lead either to reduced formation of extra-cellular collagen fibers or to an accumulation of fibers with altered properties. Unfortunately, only a few inhibitors of collagen synthesis are available, despite the importance of this protein in sustaining tissue integrity and its involvement in various disorders.
For example, cytotoxic drugs have been used in an attempt to slow the proliferation of collagen-producing fibroblasts [J. A. Casas, et al., Ann. Rhem. Dis., Vol. 46. p. 763 (1987)], such as colchicine, which slows collagen secretion into the extracellular matrix [D. Kershenobich, et al., N. Engl. J. Med., Vol. 318, p. 1709 (1988)], as well as inhibitors of key collagen metabolism enzymes (K. Karvonen, et al., J. Biol. Chem., Vol. 265, p. 8414 (1990); C. J. Cunliffe, et al., J. Med. Chem., Vol. 35, p.2652 (1992)].
Unfortunately, none of these inhibitors are collagen-type specific. Also, there are serious concerns about the toxic consequences of interfering with biosynthesis of other vital collagenous molecules, such as Clq in the classical complement pathway, acetylcholine esterase of the neuro-muscular junction endplate, conglutinin and pulmonary surfactant apoprotein.
Other drugs which can inhibit collagen synthesis, such as nifedipine and phenytoin, inhibit synthesis of other proteins as well, thereby non-specifically blocking the collagen biosynthetic pathway [T. Salo, et al., J. Oral Pathol. Med., Vol. 19, p. 404 (1990)].
Collagen cross-linking inhibitors, such as β-amino-propionitrile, are also non-specific, although they can serve as useful anti-fibrotic agents. Their prolonged use causes lathritic syndrome and interferes with elastogenesis, since elastin, another fibrous connective tissue protein, is also cross-linked. In addition, the collagen cross-linking inhibitory effect is secondary, and collagen overproduction has to precede its degradation by collagenase. Thus, a type-specific inhibitor of the synthesis of collagen itself is clearly required as an anti-fibrotic agent.
Such a type-specific collagen synthesis inhibitor is disclosed in U.S. patent application Ser. No. 08/181,066 for the treatment of a fibrotic condition, restenosis or glomerulo-sclerosis. This specific inhibitor is a composition with a pharmaceutically effective amount of a pharmaceutically active compound of a formula: wherein:                n is 1 or 2        R1 is a member of the group consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;        R2 is a member of the group consisting of hydroxy, acetoxy and lower alkoxy, and        R3 is a member of the group consisting of hydrogen and lower alkenoxy-carbonyl. Of this group of compounds, Halofuginone has been found to be particularly effective for such treatment.        
U.S. Pat. No. 5,449,678 discloses that these compounds are effective in the treatment of fibrotic conditions such as scleroderma and GVHD. WO No. 96/06616 further discloses that these compounds are effective in treating restenosis. The two former conditions are associated with excessive collagen deposition, which can be inhibited by Halofuginone. Restenosis is characterized by smooth muscle cell proliferation and extracellular matrix accumulation within the lumen of affected blood vessels in response to a vascular injury [Choi et al, Arch. Surg., Vol. 130, p. 257-261 (1995)]. One hallmark of such smooth muscle cell proliferation is a phenotypic alteration, from the normal contractile phenotype to a synthetic one. Type I collagen has been shown to support such a phenotypic alteration, which can be blocked by Halofuginone [Choi et al., Arch. Surg., Vol. 130, p. 257-261 (1995); U.S. Pat. No. 5,449,678]. Thus, Halofuginone can prevent such differentiation of smooth muscle cells after vascular injury by blocking the synthesis of type I collagen. Other in vitro studies show that Halofuginone can also inhibit the proliferation of 3T3 fibroblast cells [U.S. Pat. No. 5,449,678].
However, the in vitro action of Halofuginone does not always predict its in vivo effects. For example, Halofuginone inhibits the synthesis of collagen type I in bone chrondrocytes in vitro, as demonstrated in U.S. Pat. No. 5,449,678. However, chickens treated with Halofuginone were not reported to have an increased rate of bone breakage, indicating that the effect is not seen in vivo. Thus, the exact behavior of Halofuginone in vivo cannot always be predicted from in vitro studies.
Furthermore, the ability of Halofuginone or other related quinolinones to block or inhibit physiological processes related to tumor growth and progression is not known in the prior art. Although Halofuginone has been shown to have a specific inhibitory effect on the synthesis of type I collagen, such inhibition has not been previously shown to slow or halt tumor progression, particularly in vivo.
There is thus a widely recognized unmet medical need for an inhibitor of tumor progression which is particularly effective in vivo, substantially without adversely affecting other physiological processes.